Prion diseases are disorders of protein conformation. The pathogenic form of the prion protein, PrP/sc, is distinguished from its cellular counterpart, PrP/C, by a marked increase in the degree of beta-sheet content and a decrease in the proportion of alpha-helix. Although progress has been made in efforts to determine the three-dimensional structure of PrP/c, considerable work remains. Even more challenging is determining the structure of PrP/sc, which is extremely insoluble in the native state. The insolubility of PrP/sc has hindered attempts to elucidate the complete molecular structure of the infectious prion. To gain insight into the structural transitions which feature in PrP/sc formation, we plan to create mutations in PrP which will facilitate structural studies of PrP/sc. Our goal is to create novel mutations which lower the activation energy barrier for spontaneous formation of infectious prions de novo, especially using recombinant PrP in an in vitro reaction. By using computational methods, we have shown that it is possible to design highly pathogenic mutations in PrP which cause disease with unprecedented rapidity. Preliminary studies indicate that transmissible prions may be formed de novo, and spectroscopic studies have shown that the mutant protein adopts a beta-sheet conformation under conditions where the wild- type protein is predominantly alpha-helical. Of major importance, the beta-sheet conformer formed in vitro is soluble and preliminary NMR studies have been initiated. In a complementary approach, we have identified a mutated PrP/c molecule of 106 residues that can be converted into a protein that closely resembles PrP/sc when expressed in scrapie- infected mammalian cells. This truncated PrP/sc-like molecule is soluble in the presence of low concentrations of ionic detergent, facilitating purification of the protein for structure determination. In this proposal, we describe experimental studies which exploit these findings and extend out knowledge or PrP/sc formation. Such investigations in concert with those outlined in Projects 2, 3 and 4 should allow us to determine the complete molecular structure for a prion.